We are requesting continuing support for years 04-09. Our accomplishments during the previous funding period include studying the role of HSV amplicon mediated chemokine delivery alone or in combination with the co-stimulatory ligand CD40L in the murine pre B-cell tumor model A20, and the CT26 adenocarcinoma model. We demonstrated the utility of transducing tumors with the chemokines SLC alone and in combination with CD40L in eliciting recruitment of naive T-cells and dendritic cells, and facilitating development of a systemic adaptive immune response. We also demonstrated the potential utility of helper virus free HSV amplicons for transduction of human lymphoid malignancies. HSV amplicons encoding chemokines and/or costimulatory ligands elicit an unusually vigorous response resulting in eradication of multicentric tumors and the establishment of long lasting immunity. This suggests a particularly productive interplay between the innate and adaptive immune systems. We propose to continue studies of HSV amplicon mediated immunotherapy in murine tumors and human lymphoid tumors, with new emphasis on the role of the innate immune response. The overall goal is to understand the nature of the innate response to HSV amplicon transduction, and the role of the innate response in priming a subsequent adaptive response. Aim I, will focus on HSV amplicon engagement of the innate immune system. The chemokine/cytokine profile generated in response to vector transduction in normal splenocytes in vitro will be studied. We will study TLR receptor signaling in response to HSV amplicons. In Aim II we will compare the anti-tumor response mediated by SLC delivered using HSV amplicon vectors to that seen with nonviral delivery systems. Next we will study the effect of HSV amplicons encoding effector molecules that will selectively recruit and activate the innate system on anti-tumor response. HSV amplicons encoding chemokines that can recruit NK cells and/or NKG2D ligands will be delivered to augment the innate response in the mouse tumor models CT26, and A20. Using Ovalbumin expressing EG.7 cells, and OVA specific TCR transgenic T-cells from OT-1 mice, we will study how signals from within the innate system impact subsequent development of an adaptive response. Mice with genetic defects in key components of the innate response and the innate/adaptive transition will be tested for ability to support OT-1 activation and expansion following amplicon transduction. In Aim III we will study HSV amplicon capacity to induce NKG2D ligands on human Chronic Lymphocytic Leukemia (CLL) cells. We will also assay effects of direct transduction using HSV amplicons encoding NKG2D ligands on the immunogenicity of CLL cells. Insights gained from murine and human studies proposed will afford a better understanding of the innate contribution to development of an adaptive anti tumor response, and may lead to novel clinical approaches.